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Cholera Toxin Assault on Lipid Membranes Containing Ganglioside Gm1: an X-Ray Reflectivity and Grazing Incidence Diffraction Study at the Air-Water Interface

Chad E. Miller1, Jaroslaw Majewski1, Erik B. Watkins1, Kristian Kjaer2, and Tonya L. Kuhl3. (1) Los Alamos Neutron Science Center, Los Alamos National Laboratory, Manuel Lujan Jr. Neutron Scattering Center, TA 53, Bld 622, MS H805, Los Alamos, NM 87545, (2) Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark, (3) Chemical Engineering and Materials Science, University of California at Davis, 1 Shields Avenue, Davis, CA 95616

Cholera toxin (CTAB5) is highly efficient in taking over host organisms. To reap its destructive effects on the cell, cholera toxin must bind to and infiltrate the cellular membrane, a specialized and controlled barrier. The mechanism by which cholera toxin crosses the membrane remains unresolved. Using x-ray reflectivity, we were able to monitor the binding of cholera toxin, show that protein coverage was near maximum occupancy, and follow the penetration of the cholera protein into the lipid monolayer at the air-water interface. Protein penetration in the absence of cholera toxin's A subunit at pH=8 may suggest that the B5 pentamer (CTB5) plays a more active role in the membrane penetration mechanism than solely binding cholera toxin to its cell surface receptor. Grazing incidence x-ray diffraction (GIXD) revealed a decrease in the in-plane and out-of-plane order of the model lipid membrane after protein binding with an additional perturbation after protein activation. In addition to disrupting the order, cholera toxin binding also caused an increase in tilt of the lipid molecules with a commensurate thinning of the monolayer. Furthermore, the largest perturbation to the monolayer was caused by the full toxin at low pH, supporting the important role of low endosomal pH in the infection pathway.

Utilizing GIXD, the structure of cholera toxin (CTAB5) bound to its putative ganglioside receptor has been studied. This is one of very few proteins to be characterized in two dimensions in a fully hydrated state. The observed GIXD Bragg peaks indicate cholera toxin has a hexagonal 2D unit cell. The pentameric binding portion of cholera toxin (CTB5), in particular at low pH, had improved in-plane ordering over the full toxin (CTAB5). Enzymatically cleaving the A subunit (activation of the full toxin) also increased the protein layer ordering. These findings are consistent with A-subunit flexibility and motion causing packing inefficiencies and greater disorder of the protein layer. Corroborative evidence was provided by Bragg rod analysis, which indicated a shortening of the scattering units in the cholera layer after activation of CTAB5. These studies revealed key changes in the behavior of the cholera toxin-lipid system under different pH conditions.